The overall objective of this project is to help clarify the exact biochemical sequence of events which occurs on Beta-adrenergic stimulation of protein secretion from the parotid gland. Current evidence suggests that cAMP,Ca++, calmodulin, and protein phosphorylation probably play important roles in controlling this secretion, and the specific purpose of this project is to determine insofar as possible whether or not any of these agents act by modulating the fusion of the secretion granules to the plasma membrane which takes place during exocytosis and, if so, to determine the way in which they accomplish this. Since work with other tissues has suggested that GTP and SH groups may be involved in the secretion granule-plasma membrane interaction, we will also attempt to determine if this may likewise be the case in the rat parotid gland. In order to test for involvement of these factors in secretion granule- plasma membrane fusion, we will examine the effects of cAMP, GTP and its analogues, Ca++, calmodulin, SH-binding reagents, SH-oxidizing agents, S-S reducing agents, and phosphorylation of secretion granule and plasma membrane proteins, on the binding of (125)I-labeled plasma membrane vesicles to secretion granules and, if indicated, on the increase in octadecylrhodamine B (R18) fluorescence when R18-loaded plasma membrane vesicles fuse with secretion granules. We will also attempt to identify the membrane proteins involved in the binding and fusion, and test the effects of the same agents on their interaction; determine if the protein phosphorylation we have observed occurs during fusion and if it is catalyzed by calmodulin-, phospholipid-, and/or cAMP-dependent protein kinases; identify the plasma membrane and secretion granule proteins which contain SH groups and those which bind calmodulin, GTP and/or cAMP, and determine if any of these proteins are the ones which interact during fusion and/or serve as substrates for the protein kinases. These studies should help clarify the sequence of events in Beta- adrenergic stimulation of protein secretion by the parotid gland, a process which is important in maintenance of oral health. It may also provide information of significance in understanding secretion by other glands as well. This information in turn would provide a baseline for subsequent analysis of the exact defect which occurs in pathological failure of limitation of secretion by salivary glands and in diseases of other glands as well.